Deciphering microbial mechanisms for hexavalent chromium reduction in contaminated sediment and paddy soil

Deciphering microbial mechanisms for hexavalent chromium reduction in contaminated sediment and paddy soil | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Deciphering microbial mechanisms for hexavalent chromium reduction in contaminated sediment and paddy soil Xin Zhang, Zexin Wang, Muhammad Usman Ghani, Tianle Kong, Weimin Sun, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8418132/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hexavalent chromium [Cr(VI)] is a heavy metal that poses serious environmental risk. Microbial Cr(VI) reduction is a potential remediation approach to reduce the mobility and toxicity of this metal in the environment. However, the diversity and metabolic mechanisms of Cr(VI)-reducing bacteria (CrRBs) remain unknown. In this study, a combination of enrichment incubation and high-throughput sequencing was used to elucidate CrRBs and their associated metabolic pathways for Cr(VI) reduction. Enrichment incubation identified bacterial populations belonging to Cellulomonas, Enterobacter, Rikenellaceae , and Citrifermentans as putative CrRBs in the two Cr(VI)-contaminated sediments and paddy soils as they gradually dominated the microbial community. High-quality metagenome-assembled genomes (MAGs) associated with putative CrRBs were reconstructed, and functional genes responsible for Cr(VI) reduction were detected, suggesting that they are putative CrRBs. This study advances our understanding of CrRBs diversity and their underlying metabolic mechanisms. Chromate reduction chromium-reducing bacteria microbial diversity microbial mechanism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Chromium (Cr) is a toxic metal that is extensively distributed in the Earth’s crust and has an approximate concentration of 100 mg kg − 1 (Kazakis et al., 2015 ; Zhao et al., 2023 ). Due to its extensive use in industries, including the manufacture of refractory materials, production of chemical color pigments, and chrome plating, large amounts of Cr are released into the environment, resulting in the severe contamination of soil and water (Rahman and Thomas, 2021 ). Therefore, remediation of Cr contamination has attracted significant attention. Cr occurs in nature in different valence states ranging from -II to + VI. The two dominant species in the environment are Cr(VI) and Cr(III) (Zhong et al., 2017 ). Of these two states, Cr(VI) is extremely toxic and exhibits high mobility, whereas Cr(III) is less toxic and tends to form insoluble minerals at neutral pH values (Zhang et al., 2020 ; Bishop et al., 2019 ). Therefore, the reduction of Cr(VI) to Cr(III) contributes to the remediation of Cr-contaminated sites and rehabilitation of environments affected by Cr contamination. Microbially mediated Cr(VI) reduction is an efficient remediation strategy that has attracted the interest of the scientific community due to its cost-effectiveness and environmental sustainability (Le et al., 2023 ; Min et al., 2024 ). In microbial Cr(VI) reduction, electron donors play an important role in the biogeochemical process, and various electron donors have been used, such as organic matter, CH 4 , Fe(0), S(0), and H 2 (Lara et al., 2017 ; He et al., 2020 ; Lai et al., 2016 ; Long et al., 2017 ; Shi et al., 2019 ). In addition, several Cr(VI)-reducing bacteria (CrRBs) have been isolated from various habitats, including Enterobacter cloacae HO1 (Wang et al., 1989 ), Cellulomonas tarbata (Viti et al., 2003 ), Pseudomonas sp. G1DM21 (Desai et al., 2008 ), Bacillus cereus (He et al., 2010 ), Lysinibacillus sp. (Chen et al., 2021 ), and Lactococcus lactis AM99 (Akhzari et al., 2024 ). Most of these bacteria are heterogeneous because organics can support high microbial activity, making heterotrophic Cr(VI) reduction a promising remediation technology. However, the potential use of Cr(VI)-reducing bacteria, particularly indigenous microbial communities, in the remediation of Cr(VI)-contaminated environments may be underestimated because of the inherent limitations of culture-dependent analytical techniques. Most studies have focused on identifying CrRBs and evaluating their chromate transport capability. For instance, Anoxybacillus flavithermus ABF1 was isolated as an alkaline Cr(VI)-reducing bacterium from a geothermal region, and was able to reduce 93.71% of Cr(VI) at an initial concentration of 20 mg L − 1 in 72 hours (Yang et al., 2022 ). Members of the Comamonadaceae family have been identified in methane- and oxygen-based membrane biofilm reactor with the potential for chromate reduction, which could reduce Cr(VI) to Cr(III) at a rate of 89% (Long et al., 2017 ). Zhang et al. ( 2020 ) proposed a mechanism for the metabolic pathway of Cr(VI)-reducing bacteria in a sulfur-based mixotrophic system by determining the abundance of the chrA genes and the compounds involved in Cr(VI) reduction (Zhang et al., 2020 ). However, the reduction mechanisms of most Cr(VI)-reducing bacteria are not clearly understood because most of them have not been isolated, resulting in a lack of molecular biological evidence. Therefore, a better understanding of the metabolic mechanisms of Cr(VI)-reducing bacteria is required. In this study, anaerobic cultures were established to enrich indigenous Cr(VI)-reducing bacteria using long-term Cr(VI)-contaminated sediments and paddy soils as inocula. A metagenomic approach was used to reconstruct the metagenome-assembled genomes (MAGs) of CrRBs from these complex microbial communities and predict the Cr(VI) reduction pathway. The aim of the current study was to investigate (i) which microorganisms are responsible for Cr(VI)-reducing bacteria in Cr(VI)-contaminated sediments and paddy soils and (ii) the metabolic mechanisms of CrRBs. These findings provide new insights into the diversity and metabolic potential of CrRBs for Cr(VI) reduction, which may contribute to the remediation of Cr(VI) in contaminated environments. 2. Materials and Methods 2.1 Sample collection and characterization Samples were collected from river sediments at depth of 0–20 cm in Xikuangshan, Hunan, China (27°44′ N, 111°28′ E, designated as XKS), and from flooded rice fields in Wanshan, Guizhou, China (27°31′ N, 109°12′ E, designated as WS), both located near the mining area. The collected samples were immediately transported to the laboratory on ice and stored at -20 ℃ for chemical analysis and microcosm incubation. To determine the Cr content, the samples were freeze-dried, digested with an HCl: HNO 3 solution (3:1, v/v), and measured by ICP-OES (Vista MPX, Varian). The samples of XKS and WS contained 237.3 mg kg − 1 and 136.8 mg kg − 1 of Cr, respectively. 2.2 Anaerobic enrichment cultures Anaerobic enrichment cultures were established using the collected samples to detect potential Cr-reducing bacteria. Approximately 2 g of the sample was added to 100 mL of mineral salt medium (MSM; 7.9 g L − 1 Na 2 HPO 4 ·7H 2 O, 1.5 g L − 1 KH 2 PO 4 , 0.3 g L − 1 NH 4 Cl, 0.1 g L − 1 MgSO 4 ·7H 2 O, 10 mL L –1 vitamin solution, and 5 mL L –1 trace element solution) as inoculum (Zhang et al., 2015 ). The bottles were then purged with N 2 for 30 min, capped with butyl rubber septa and aluminum caps, and inoculated for three days at room temperature to degrade soluble organics and indigenous Cr in the original inoculum. Cr(VI) was added to the cultures at a final concentration of 20 mg L − 1 and 6 mM sodium acetate was used as the electron donor. In addition, abiotic controls were prepared using autoclaved samples to demonstrate that the biological activity mediates this process. All cultures were prepared in triplicates at each sampling time point and incubated at 30 o C. During incubation, the Cr(VI) concentration was monitored by taking 1 mL of supernatant from each culture for colorimetric measurement according to the established protocol (He et al., 2020 ). After the complete removal of Cr(VI), the cultures were spiked again with 20 mg L − 1 of Cr(VI) (which was considered as one cycle), and duplicate cultures were destructively sampled for microbial community analysis. Three samples were analyzed for each case. 2.3 DNA extraction Cultures collected from each enrichment cycle were centrifuged at 7,000 rpm for 10 min. Total genomic DNA was extracted from the cultures and raw samples using the DNeasy PowerSoil DNA Kit (QIAGEN, Germany) according to the manufacturer’s protocol. DNA was not extracted from the sterile controls in this study. 2.4 Illumina MiSeq sequencing of 16S rRNA gene amplicons To characterize the prokaryotic communities, the hypervariable region V4 of the 16S rRNA gene was amplified using extracted DNA as a template. The forward primer was 515F (5’-GTGYCAGCMGCCGCGGTAA-3’) and the reverse primer was 806R (5’-GGACTACNVGGGTWTCTAAT-3’) (Klindworth et al., 2013 ). The amplification products were sequenced on an Illumina MiSeq platform at Personal Biotechnology (Shanghai, China). The raw amplicon sequencing data were analyzed using the QIIME2-202002 toolkit (Bolyen et al., 2019). Briefly, raw reads were merged, trimmed, and filtered. The filtered reads were clustered into amplicon sequence variants (ASV) using DADA2 (Callahan et al., 2016 ). Representative sequences were assigned to the SILVA 132 database (Quast et al., 2012 ). 2.5 Shotgun metagenomic sequencing and analysis In the third cycle, DNA from triplicate cultures was combined to create a composite DNA sample because the amount of DNA extracted from each culture was insufficient for shotgun metagenomic sequencing. The DNA samples were then sequenced on an Illumina HiSeq 4000 platform (PersonalBio, Shanghai, China). Approximately 33,934,255 and 33,985,822 raw reads were generated from the XKS and WS metagenomes, respectively. All raw reads were trimmed using Trimmomatic 0.39 (Bolger et al., 2014 ) with the following arguments: “sliding window: 4:15, leading: 3, trailing: 3, and minilen: 80”. Overlapping read pairs were identified and merged using FLASH with a minimum overlap of 15 bp and a maximum overlap of 100 bp. Clean datasets were assembled de novo into contigs using MEGAHIT (Li et al., 2015 ) (k = 21–141, step = 10). Metagenome-assembled genomes (MAGs) were retrieved from the assembled contigs by metagenome binning using MaxBin2 (Wu et al., 2016 ), MetaBAT2 (Kang et al., 2015 ), and CONCOCT (Alneberg et al., 2014 ). The obtained MAGs were refined and their quality was improved using DAS (Sieber et al., 2018 ). The MAGs were then filtered using an MDMcleaner (Vollmers et al., 2022 ) and their quality was assessed using CheckM (Parks et al., 2015 ). Only MAGs with a completion rate of > 60% and contamination rate of < 10% were retained and subjected to taxonomic annotation with GTDB-TK (Chaumeil et al., 2019 ). The functional genes of the MAGs were annotated using KofamKOALA (Aramaki et al., 2020 ) with default parameters. The phylogeny of MAGs genomes was generated using MEGA (Tamura et al., 2021 ). 2.6 Data availability Nucleic acid reads based on the 16S rRNA gene and metagenomic sequencing were submitted to the NCBI GenBank database under project number PRJNA847997. 3. Results 3.1 Microbial reduction of Cr(VI) Anaerobic enrichment cultures were established using two Cr-contaminated samples as the inocula. Cr(VI) was added for three cycles as the sole electron acceptor to enrich the Cr(VI)-reducing bacteria. The concentration of aqueous Cr(VI) was monitored during the incubation period ( Fig. 1 ) . The Cr(VI) concentration constantly decreased in the two live treatments inoculated with the XKS sediment and WS paddy soil. In contrast, the Cr(VI) concentration in the sterile control initially decreased slightly and then reached a metastable state. These observations suggest that the reduction of Cr(VI) was driven by microorganisms. Additionally, the Cr(VI) reduction rates were significantly different between the two live treatments. Approximately 20 mg L − 1 Cr(VI) was reduced to 0 mg L − 1 within 6 days in the XKS treatment, while 12 days were required for the complete reduction of Cr(VI) in the WS treatment, suggesting a higher reduction potential of Cr(VI) in the XKS treatment than in the WS treatment. The different microbial communities in the two treatments may have contributed to this outcome, or the XKS treatment may have resulted in a higher abundance of potential Cr(VI)-reducing bacteria. 3.2 Enrichment of Cr(VI) reducing bacteria (CrRB) Similar to the original inoculum, the microbial communities in both cultures were characterized at the end of each cycle. Different microbial taxa were enriched in XKS and WS cultures, as shown in Fig. 2 . At the phylum level, the relative abundance of Bacteroidota in the XKS cultures gradually increased during the incubation period and reached the level of the dominant population (36%) after three cycles. At the same time, the relative abundance of Desulfobacterota in the XKS cultures increased from 2% to 16%. During incubation, the relative abundances of Actinobacteriota and Proteobacteria in the WS cultures were 7% and 11%, respectively, whereas those in the original inoculum were 42% and 36%, respectively. Additionally, Proteobacteria and Firmicutes were the dominant bacteria in the XKS and WS cultures, accounting for 64% and 65% of the total abundance, respectively, while their abundance decreased to 18% and 13%, respectively, at the end of incubation. These findings suggest that distinct microbial communities mediate Cr(VI) reduction and reveal that Cr(VI) exerts a strong selective pressure on different microorganisms. At the genus level, uncultured Rikenellaceae became the most abundant taxon in the XKS enrichment cultures, with a relative abundance of 21% in the third cycle compared to 0.4% in the original inoculum, followed by Citrifermentans with a relative abundance of 7% ( Fig. 3 ) . After three cycles of incubation, bacteria associated with Cellulomonas and Enterobacter gradually dominated, with relative abundances of 36% and 28%, respectively, in the WS cultures. In addition, the relative abundances of other genera, such as Thiobacillus , Brevundimonas , Clostridium , and Desulfitobacterium , decreased with incubation time. These findings suggest that uncultured Rikenellaceae , Citrifermentans , Cellulomonas , and Enterobacter may be responsible for Cr(VI) reduction. 3.3 Metagenomic analysis of CrRBs Metagenomic binning was performed to retrieve genomic information on the proposed CrRBs and further elucidate their metabolic potential. A total of 21 metagenomic-assembled genomes (MAGs) with > 60% completeness and < 10% contamination were obtained from the two metagenomes. These MAGs were categorized into seven different bacterial phyla: Halobacteriota , Acidobacteriota , Chloroflexota , Bacteroidota , Proteobacteria , Desulfobacterota , and Actinobacteriota ( Fig. 4 ) . Among the recovered MAGs, seven were taxonomically linked to putative CrRB, including Rikenellaceae_uncultured (bin.5 and bin.8), Citrifermentans (bin.9 and bin.11), Cellulomonas (bin.4 and bin.5) and Enterobacter (bin.1), which were selected for further investigation. Functional genes of MAGs associated with CrRBs were identified ( Fig. 5 ) . Key genes involved in chromate reduction ( chrR ) and chromate transport ( chrA ) (He et al., 2018; Lu et al., 2020) were detected in all putative CrRB-associated MAGs except for Cellulomonas -associated MAGs. In addition, other genes potentially involved in Cr(VI) reduction have been examined in these MAGs, including ssuE , azoR , rutE , nfsA , ribF , nemA , nrfA , and modA (Xia et al., 2018 ; Xia et al., 2021 ). These observations suggested that these bacteria have the potential to reduce Cr(VI). In addition, putative CrRB-associated MAGs harbored diverse genes related to metal resistance, such as czcABCD , arsC, arsR, cusB, and znuABC, suggesting their genetic potential to adapt to metal-contaminated habitats. All seven CrRB-associated MAGs harbored genes encoding for six major carbon fixation pathways, indicating their ability to grow with acetic acid. In addition, some genes involved in nitrogen and sulfur metabolism were detected in the CrRB-associated MAGs. 4. Discussion Microbial-driven Cr(VI) reduction has important environmental implications for the remediation of Cr-contaminated environments and has been widely studied in natural habitats, such as soils (Hou et al., 2020 ; Fu et al., 2021 ) and groundwater (Shi et al., 2020 ). In this study, the diversity and metabolic potential of Cr(VI)-reducing microbial communities in sediments and paddy soils were investigated using anaerobic enrichment cultures and metagenomics. 4.1 Potential Cr(VI)-reducing bacteria Biological Cr(VI) reduction has been observed in the cultures inoculated with Cr-contaminated sediment and paddy soil from two sites whose aqueous Cr(VI) reduced in 6 and 12 days, respectively. Four bacteria, Rikenellaceae , Citrifermentans , Cellulomonas , and Enterobacter , were identified as putative CrRBs based on their relative abundances in the cultures, which increased significantly with incubation time. After incubation, the dominant taxa in the cultures among these CrRBs were Rikenellaceae , Cellulomonas , and Enterobacter , with relative abundances of more than 20%. Anaerobic fermentation bacteria associated with the Rikenellaceae family have been found in anaerobic environments including swamps (Su et al., 2014 ) and anaerobic activated sludge (Yan et al., 2021 ). Some studies have shown that members of the Rikenellaceae are enriched during the decomposition and digestion of organic matter (Tian et al., 2019 ; Chen et al., 2022 ), suggesting that they may have the ability to utilize acetate. It has been reported that Rikenellaceae are potentially syntrophic bacteria that can directly transfer electron with methanogens through iron oxide minerals (Lee et al., 2019 ). In addition, slight enrichment of Rikenellaceae was observed in the reduction of Cr(VI) and nitrate driven by the microbiome and iron oxide minerals (Hu et al., 2021 ), suggesting that Rikenellaceae may have the potential to reduce or tolerate Cr(VI). However, the ability of Rikenellaceae spp. to reduce Cr(VI) has not yet been reported. Enterobacter spp. have been previously identified as Cr-reducing bacteria. Enterobacter cloacae HO1 was the first strain to have the ability to reduce Cr(VI) (Wang et al., 1989 ). Various members of Enterobacter have been isolated, including Enterobacter aerogenes T2 (Panda and Sarkar, 2012 ), Enterobacter cloacae B2-DHA (Rahman et al., 2015 ), and Enterobacter sp. SL (Sun et al., 2020 ). Enterobacter cloacae B2-DHA can resist 1000 mg L − 1 of Cr(VI) (Rahman et al., 2015 ), and Enterobacter sp. DU17 reduced Cr(VI) by 100 mg L − 1 over 16 h using glucose as an electron donor (Rahman and Singh, 2014 ), suggesting its strong ability to reduce Cr(VI). In addition, Cr(VI) reduction was effectively observed when Enterobacter cloacae HO1 was enriched with acetate compared to glucose, citrate, or lactate (Komori et al., 1989 ), suggesting that acetate is an efficient electron donor for Enterobacter cloacae HO1 to reduce Cr(VI). In agreement with our current study, Enterobacter dominated with increasing incubation time when acetate was used as the electron donor. Cellulomonas spp. are known to reduce Cr(VI) and some of their members have been isolated from various habitats (Brookshier et al., 2018 ; Viamajala et al., 2007 b). For example, Cellulomonas tarbata isolated from Cr-contaminated soil was resistant to Cr(VI) concentrations up to 12 mM (Viti et al., 2003 ). In another study, Cellulomonas WS01 reduced 0.2 mM of Cr(VI) in 70 hours (Sani et al., 2002 ). These results indicated that bacteria associated with Cellulomonas can reduce Cr(VI). Additionally, Cellulomonas ES6 was isolated from Cr-contaminated sediment reduced anaerobically with Cr(VI) using acetate as an electron donor (Viamajala et al., 2007 b), similar to the conditions used in this study. Citrifermentans are a rarely studied group of anaerobic bacteria and only three strains have been isolated from this genus: Citrifermentans bemidjiense (Nevin et al., 2005 ), Citrifermentans bremense , and Citrifermentans pelophilum (Straub and Buchholz-Cleven, 2001 ). These three bacteria have been previously identified as Fe(III)-reducing bacteria (Straub and Buchholz-Cleven, 2001 ; Aklujkar et al., 2010 ; Neal et al., 2004 ), indicating their potential for metal reduction. Although some members of the Geobacteraceae family have been reported to reduce Cr(VI) (Shi et al., 2019 ; He et al., 2019 ), identification of the genus Citrifermentans has not yet been reported. 4.2 Metabolic Potentials of CrRB-Associated MAGs The genetic traits responsible for Cr(VI) reduction are of particular significance as they may represent some of the most significant characteristics for the bioremediation of Cr. Metagenomic binning was employed to retrieve bins associated with putative Cr(VI)-reducing bacteria, thereby enabling the exploration of the underlying mechanisms involved in Cr(VI) reduction. Various gene families hypothesized to be involved in Cr(VI) reduction were selected to examine the presence of these genes within the MAGs linked to putative Cr(VI)-reducing bacteria. C hrR , which belongs to the flavodoxin superfamily, is a chromate reductase (Eswaramoorthy et al., 2012 ). A reductase in the NADPH-dependent flavoprotein family, ChrA, also plays a key role in Cr(VI) reduction and resistance (Ramirez-Diaz et al., 2008 ). During reduction, ChrR transfers NADH electrons to Cr(VI) and directly reduces Cr(VI), whereas ChrA serves as a chromate transporter that exports Cr(III) into the extracellular environment. In this study, chrR was detected in MAGs associated with Citrifermentans and Enterobacter , whereas chrA was detected in MAGs from Rikenellaceae , Citrifermentans , and Enterobacter , suggesting that either or both enzymes may be used to reduce Cr(VI). Neither chrR nor chrA were detected in Cellulomonas -associated MAGs, which may be attributed to incomplete metagenomic binning that did not provide the whole genomes of these bacteria. The genomes of the Cellulomonas sp. strains K38 and B12 were annotated using chromate reductases (ChrRs) (Brookshier et al., 2018 ). In addition to chrR and chrA , Cr(VI) reduction has been observed to be mediated by flavoprotein reductase (RibF) in Cr(VI)-reducing Serratia sp. S2 (Dong et al., 2018 ). This gene was observed in all the putative CrRB-associated MAGs. Some genes have been reported to be responsible for Cr(VI) reduction in various bacteria including nrfA , ssuE , rutE , azoR , nemA , and nfsA (Yang et al., 2022 ; Bhattacharya et al., 2015 ; Chen et al., 2021 ; Xia et al., 2018 ). These genes were detected in putative CrRB-associated MAGs, indicating that putative CrRBs may use various pathways for Cr(VI) reduction. These bacteria were further verified as putative Cr(VI) reducers by identifying potential functional genes for Cr(VI) reduction. In addition, putative CrRB-associated MAGs harbored several genes involved in resistance to different metals. For instance, most MAGs harbor the arsR and arsC genes, both of which are involved in As and Sb resistance and their detoxification pathways (Ji et al., 1992). Genes encoding Cd, Zn, Cu, and Mn exporters were identified in all MAGs, suggesting that CrRBs can resist or transform these metals, because the samples used in this study were contaminated with various metals. 5. Conclusions In this study, enrichment incubation and metagenomics were combined to enrich CrRBs and to elucidate the metabolic pathways involved in Cr(VI) reduction. After three enrichment cycles, Cellulomonas , Enterobacter , Rikenellaceae , and Citrifermentans dominated Cr-contaminated sediments and paddy soils, with abundances of 36, 28, 21, and 7%, respectively, indicating that these were putative CrRBs. Among these, Rikenellaceae and Citrifermentans are novel chromium-reducing bacteria. The reconstructed genomes of the four putative CrRBs encoded key genes ( chrA and chrR ) for chromate reduction, indicating their ability to reduce chromate. This study expands the known diversity of Cr(VI)-reducing bacteria and improves our understanding of their metabolism. Declarations Authors contribution Xin Zhang : Writing - Original Draft, Investigation, Data curation,Formal analysis,Methodology. Zexin Wang : Data curation,Investigation, Writing - Original Draft,Writing - Review & Editing. Muhammad Usman Ghani :Investigation,Data curation,Writing - Review & Editing. Tianle Kong :Investigation,Methodology,Validation. Weimin Sun : Formal analysis,Funding acquisition, Writing - Review & Editing. Xiaoxu Sun :Funding acquisition, Writing - Review & Editing. Baoqin Li : Visualization,Writing - Review & Editing. Zewen Tan : Data curation, Writing - Review & Editing. Geng Yan :Conceptualization,Funding acquisition,Supervision,Writing - Review & Editing. Funding This work was supported by the National Key Research and Development Program of China (Grant No. 2023YFC3706903), the National Natural Science Foundation of China (Grant Nos. U21A2035, 42321005, 42307354 and 42277247), Guangdong Basic and Applied Basic Research Foundation (Grant Nos. 2025B1515020024, 2023B1515040007, 2022B1515120033, 2024A1515011436 and 2024A1515011044), GDAS' Project of Science and Technology Development (Grant Nos. 2024GDASQNRC-0108 and 2023GDASZH-2023010104-1), China Postdoctoral Science Foundation funded Project (Grant No. 2023M740763), and Guangdong Foundation for Program of Science and Technology Research (Grant No. 2023B1212060044). Data Availability The datasets obtained during the current study are available from the corresponding author on reasonable request. Ethics Approval Not applicable. Consent for Publication This manuscript is approved by all authors for publication in Water, Air, & Soil Pollution. Competing interests The authors have no conflicts of interest to declare that are relevant to the content of this article. References Akhzari, F., Naseri, T., Mousavi, SM., Khosravi-Darani, K., 2024. A sustainable solution for alleviating hexavalent chromium from water streams using Lactococcus lactis AM99 as a novel Cr(VI)-reducing bacterium. J Environ Manage. 353, 120190. Aklujkar, M., Young, N.D., Holmes, D., Chavan, M., Risso, C., Kiss, H.E., Han, C.S., Land, M.L., Lovley, D.R., 2010. The genome of Geobacter bemidjiensis, exemplar for the subsurface clade of Geobacter species that predominate in Fe(III)-reducing subsurface environments. BMC Genomics 11, 490. Alneberg, J., Bjarnason, B.S., de Bruijn, I., Schirmer, M., Quick, J., Ijaz, U.Z., Lahti, L., Loman, N.J., Andersson, A.F., Quince, C., 2014. Binning metagenomic contigs by coverage and composition. Nat. Methods 11(11), 1144–1146. Aramaki, T., Blanc-Mathieu, R., Endo, H., Ohkubo, K., Kanehisa, M., Goto, S., Ogata, H., 2020. KofamKOALA: KEGG Ortholog assignment based on profile HMM and adaptive score threshold. Bioinformatics 36(7), 2251–2252. Bhattacharya, P., Barnebey, A., Zemla, M., Goodwin, L., Auer, M., Yannone, S.M., 2015. Complete genome sequence of the chromate-reducing bacterium Thermoanaerobacter thermohydrosulfuricus strain BSB-33. Stand. Genomic Sci. 10(1). Bishop, M.E., Dong, H., Glasser, P., Briggs, B.R., Pentrak, M., Stucki, J.W., Boyanov, M.I., Kemner, K.M., Kovarik, L., 2019. Reactivity of redox cycled Fe-bearing subsurface sediments towards hexavalent chromium reduction. Geochim. Cosmochim. Ac. 252, 88–106. Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15), 2114–2120. Bolyen, E., Rideout, J.R., Dillon, M.R., Bokulich, N.A., Abnet, C.C., Al-Ghalith, G.A., Alexander, H., Alm, E.J., Arumugam, M., Asnicar, F., Bai, Y., Bisanz, J.E., Bittinger, K., Brejnrod, A., Brislawn, C.J., Brown, C.T., Callahan, B.J., Caraballo-Rodríguez, A.M., Chase, J., Cope, E.K., Da Silva, R., Diener, C., Dorrestein, P.C., Douglas, G.M., Durall, D.M., Duvallet, C., Edwardson, C.F., Ernst, M., Estaki, M., Fouquier, J., Gauglitz, J.M., Gibbons, S.M., Gibson, D.L., Gonzalez, A., Gorlick, K., Guo, J., Hillmann, B., Holmes, S., Holste, H., Huttenhower, C., Huttley, G.A., Janssen, S., Jarmusch, A.K., Jiang, L., Kaehler, B.D., Kang, K.B., Keefe, C.R., Keim, P., Kelley, S.T., Knights, D., Koester, I., Kosciolek, T., Kreps, J., Langille, M.G.I., Lee, J., Ley, R., Liu, Y., Loftfield, E., Lozupone, C., Maher, M., Marotz, C., Martin, B.D., McDonald, D., McIver, L.J., Melnik, A.V., Metcalf, J.L., Morgan, S.C., Morton, J.T., Naimey, A.T., Navas-Molina, J.A., Nothias, L.F., Orchanian, S.B., Pearson, T., Peoples, S.L., Petras, D., Preuss, M.L., Pruesse, E., Rasmussen, L.B., Rivers, A., Robeson, M.S., Rosenthal, P., Segata, N., Shaffer, M., Shiffer, A., Sinha, R., Song, S.J., Spear, J.R., Swafford, A.D., Thompson, L.R., Torres, P.J., Trinh, P., Tripathi, A., Turnbaugh, P.J., Ul-Hasan, S., van der Hooft, J.J.J., Vargas, F., Vázquez-Baeza, Y., Vogtmann, E., von Hippel, M., Walters, W., Wan, Y., Wang, M., Warren, J., Weber, K.C., Williamson, C.H.D., Willis, A.D., Xu, Z.Z., Zaneveld, J.R., Zhang, Y., Zhu, Q., Knight, R., Caporaso, J.G., 2019. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37(9), 1091. Brookshier, A.M., Santo, D.J., Kourtev, P.S., Learman, D.R., 2018. Draft Genome Sequences of Two Bacillus sp. Strains and Four Cellulomonas sp. Strains Isolated from Heavy-Metal-Contaminated Soil. Microbiol Resour Announc 7(11). Callahan, B.J., McMurdie, P.J., Rosen, M.J., Han, A.W., Johnson, A.J.A., Holmes, S.P., 2016. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13(7), 581–583. Chai, L., Ding, C., Tang, C., Yang, W., Yang, Z., Wang, Y., Liao, Q., Li, J., 2018. Discerning three novel chromate reduce and transport genes of highly efficient Pannonibacter phragmitetus BB: From genome to gene and protein. Ecotox. Environ. Safe. 162, 139–146. Chaumeil, P., Mussig, A.J., Hugenholtz, P., Parks, D.H., 2019. GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics. Chen, J., Li, X., Gan, L., Jiang, G., Zhang, R., Xu, Z., Tian, Y., 2021. Mechanism of Cr(VI) reduction by Lysinibacillus sp. HST-98, a newly isolated Cr (VI)-reducing strain. Environ Sci Pollut Res Int 28(46), 66121–66132. Chen, J., Li, X., Gan, L., Jiang, G., Zhang, R., Xu, Z., Tian, Y., 2021. Mechanism of Cr(VI) reduction by Lysinibacillus sp. HST-98, a newly isolated Cr (VI)-reducing strain. Environ. Sci. Pollut. R. 28(46), 66121–66132. Chen, J., Yang, Y., Ke, Y., Chen, X., Jiang, X., Chen, C., Xie, S., 2022. Anaerobic sulfamethoxazole-degrading bacterial consortia in antibiotic‐contaminated wetland sediments identified byDNA ‐stable isotope probing and metagenomics analysis. Environ. Microbiol. 24(8), 3751–3763. Desai, C., Jain, K., Madamwar, D., 2008. Hexavalent chromate reductase activity in cytosolic fractions of Pseudomonas sp. G1DM21 isolated from Cr(VI) contaminated industrial landfill. Process Biochem. 43(7), 713–721. Dong, L., Zhou, S., He, Y., Jia, Y., Bai, Q., Deng, P., Gao, J., Li, Y., Xiao, H., 2018. Analysis of the Genome and Chromium Metabolism-Related Genes of Serratia sp. S2. Appl. Biochem. Biotech. 185(1), 140–152. Eswaramoorthy, S., Poulain, S., Hienerwadel, R., Bremond, N., Sylvester, M.D., Zhang, Y.B., Berthomieu, C., Van Der Lelie, D., Matin, A., 2012. Crystal structure of ChrR–a quinone reductase with the capacity to reduce chromate. PLoS One 7(4), e36017. Fu, L., Feng, A., Xiao, J., Wu, Q., Ye, Q., Peng, S., 2021. Remediation of soil contaminated with high levels of hexavalent chromium by combined chemical-microbial reduction and stabilization. J. Hazard. Mater. 403, 123847. He, C., Zhang, B., Yan, W., Ding, D., Guo, J., 2020. Enhanced Microbial Chromate Reduction Using Hydrogen and Methane as Joint Electron Donors. J. Hazard. Mater. 395, 122684. He, M., Li, X., Guo, L., Miller, S.J., Rensing, C., Wang, G., 2010. Characterization and genomic analysis of chromate resistant and reducing Bacillus cereus strain SJ1. BMC Microbiol. 10, 221. He, Y., Gong, Y., Su, Y., Zhang, Y., Zhou, X., 2019. Bioremediation of Cr (VI) contaminated groundwater by Geobacter sulfurreducens: Environmental factors and electron transfer flow studies. Chemosphere 221, 793–801. Hou, S., Wu, B., Luo, Y., Li, Y., Ma, H., Peng, D., Xu, H., 2020. Impacts of a novel strain QY-1 allied with chromium immobilizing materials on chromium availability and soil biochemical properties. J. Hazard. Mater. 382, 121093. Hu, Y., Liu, T., Chen, N., Feng, C., 2021. Iron oxide minerals promote simultaneous bio-reduction of Cr(VI) and nitrate: Implications for understanding natural attenuation. Sci. Total Environ. 786, 147396. Kang, D.D., Froula, J., Egan, R., Wang, Z., 2015. MetaBAT, an efficient tool for accurately reconstructing single genomes from complex microbial communities. PeerJ 3, e1165. Kazakis, N., Kantiranis, N., Voudouris, K.S., Mitrakas, M., Kaprara, E., Pavlou, A., 2015. Geogenic Cr oxidation on the surface of mafic minerals and the hydrogeological conditions influencing hexavalent chromium concentrations in groundwater. Sci. Total Environ. 514, 224–238. Klindworth, A., Pruesse, E., Schweer, T., Peplies, J., Quast, C., Horn, M., Glöckner, F.O., 2013. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 41(1), e1. KOMORI, K., WANG, P., TODA, K., OHTAKE, H., 1989. Factors affecting chromate reduction in Enterobacter cloacae strain HO1. Appl. Microbiol. Biot. 31(5–6), 567–570. Lai, C., Zhong, L., Zhang, Y., Chen, J., Wen, L., Shi, L., Sun, Y., Ma, F., Rittmann, B.E., Zhou, C., Tang, Y., Zheng, P., Zhao, H., 2016. Bioreduction of Chromate in a Methane-Based Membrane Biofilm Reactor. Environ. Sci. Technol. 50(11), 5832–5839. Lara, P., Morett, E., Juárez, K., 2017. Acetate biostimulation as an effective treatment for cleaning up alkaline soil highly contaminated with Cr(VI). Environ. Sci. Pollut. R. 24(33), 25513–25521. Lee, J., Koo, T., Yulisa, A., Hwang, S., 2019. Magnetite as an enhancer in methanogenic degradation of volatile fatty acids under ammonia-stressed condition. J. Environ. Manage. 241, 418–426. Yilin Le, Xing He, Mengnan Liu, Xue Liu, Shidong Zhou, Rongrong Xie, Yu Fu, Huilei Wang, Jianzhong Sun., 2023. Light-driven sustainable enhancement of Cr(VI) reduction via the combination of Cr(VI)-reducing bacteria, Paraclostridium bifermentans with CdS nanoparticles, Journal of Environmental Chemical Engineering, J. Environ. Chem. Eng. 11(5),110364. Li, D., Liu, C., Luo, R., Sadakane, K., Lam, T., 2015. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics 31(10), 1674–1676. Long, M., Zhou, C., Xia, S., Guadiea, A., 2017. Concomitant Cr(VI) reduction and Cr(III) precipitation with nitrate in a methane/oxygen-based membrane biofilm reactor. Chem. Eng. J. 315, 58–66. Magoc, T., Salzberg, S.L., 2011. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27(21), 2957–2963. Min, XY., Zhang, KJ., Jianxin Chen, Liyuan Chai, Zhang Lin, Long Zou, Weizao Liu, Chunlian Ding, Yan Shi., 2024. Bacteria-driven copper redox reaction coupled electron transfer from Cr(VI) to Cr(III): A new and alternate mechanism of Cr(VI) bioreduction. J HAZARD MATER. 462, 132485. Neal, A.L., Clough, L.K., Perkins, T.D., Little, B.J., Magnuson, T.S., 2004. In situ measurement of Fe(III) reduction activity of Geobacter pelophilus by simultaneous in situ RT-PCR and XPS analysis. FEMS Microbiol. Ecol. 49(1), 163–169. Nevin, K.P., Holmes, D.E., Woodard, T.L., Hinlein, E.S., Ostendorf, D.W., Lovley, D.R., 2005. Geobacter bemidjiensis sp. nov. and Geobacter psychrophilus sp. nov., two novel Fe(III)-reducing subsurface isolates. Int J Syst Evol Microbiol 55(Pt 4), 1667–1674. Panda, J., Sarkar, P., 2012. Bioremediation of chromium by novel strains Enterobacter aerogenes T2 and Acinetobacter sp. PD 12 S2. Environ. Sci. Pollut. R. 19(5), 1809–1817. Parks, D.H., Imelfort, M., Skennerton, C.T., Hugenholtz, P., Tyson, G.W., 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 25(7), 1043–1055. Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., Glöckner, F.O., 2012. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41(D1), D590-D596. Rahman, A., Nahar, N., Nawani, N.N., Jass, J., Hossain, K., Saud, Z.A., Saha, A.K., Ghosh, S., Olsson, B., Mandal, A., 2015. Bioremediation of hexavalent chromium (VI) by a soil-borne bacterium, Enterobacter cloacae B2-DHA. J Environ Sci Health A Tox Hazard Subst Environ Eng 50(11), 1136–1147. Rahman, A., Nahar, N., Nawani, N.N., Jass, J., Hossain, K., Saud, Z.A., Saha, A.K., Ghosh, S., Olsson, B., Mandal, A., 2015. Bioremediation of hexavalent chromium (VI) by a soil-borne bacterium, Enterobacter cloacae B2-DHA. Journal of environmental science and health. Part A, Toxic/hazardous substances & environmental engineering 50(11), 1136–1147. Rahman, Z., Singh, V.P., 2014. Cr(VI) reduction by Enterobacter sp. DU17 isolated from the tannery waste dump site and characterization of the bacterium and the Cr(VI) reductase. Int. Biodeter. Biodegr. 91, 97–103. Rahman, Z., Thomas, L., 2021. Chemical-Assisted Microbially Mediated Chromium (Cr) (VI) Reduction Under the Influence of Various Electron Donors, Redox Mediators, and Other Additives: An Outlook on Enhanced Cr(VI) Removal. Front. Microbiol. 11. Ramirez-Diaz, M.I., Diaz-Perez, C., Vargas, E., Riveros-Rosas, H., Campos-Garcia, J., Cervantes, C., 2008. Mechanisms of bacterial resistance to chromium compounds. Biometals 21(3), 321–332. SANI, R.K., PEYTON, B.M., SMITH, W.A., APEL, W.A., PETERSEN, J.N., Idaho, N.L.I., 2002. Dissimilatory reduction of Cr(VI), Fe(III), and U(VI) by Cellulomonas isolates. Appl. Microbiol. Biot. 60(1–2), 192–199. Shi, C., Cui, Y., Lu, J., Zhang, B., 2020. Sulfur-based autotrophic biosystem for efficient vanadium (V) and chromium (VI) reductions in groundwater. Chem. Eng. J. 395, 124972. Shi, J., Zhang, B., Qiu, R., Lai, C., Jiang, Y., He, C., Guo, J., 2019. Microbial Chromate Reduction Coupled to Anaerobic Oxidation of Elemental Sulfur or Zerovalent Iron. Environ. Sci. Technol. 53(6), 3198–3207. Sieber, C.M.K., Probst, A.J., Sharrar, A., Thomas, B.C., Hess, M., Tringe, S.G., Banfield, J.F., 2018. Recovery of genomes from metagenomes via a dereplication, aggregation and scoring strategy. Nature Microbiology 3(7), 836–843. Straub, K.L., Buchholz-Cleven, B.E., 2001. Geobacter bremensis sp. nov. and Geobacter pelophilus sp. nov., two dissimilatory ferric-iron-reducing bacteria. Int J Syst Evol Microbiol 51(Pt 5), 1805–1808. Su, X.L., Tian, Q., Zhang, J., Yuan, X.Z., Shi, X.S., Guo, R.B., Qiu, Y.L., 2014. Acetobacteroides hydrogenigenes gen. nov., sp. nov., an anaerobic hydrogen-producing bacterium in the family Rikenellaceae isolated from a reed swamp. Int J Syst Evol Microbiol 64(Pt 9), 2986–2991. Sun, Y., Lan, J., Du, Y., Guo, L., Du, D., Chen, S., Ye, H., Zhang, T.C., 2020. Chromium(VI) bioreduction and removal by Enterobacter sp. SL grown with waste molasses as carbon source: Impact of operational conditions. Bioresource Technol. 302, 121974. Tamura, K., Stecher, G., Kumar, S., 2021. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 38(7), 3022–3027. Tian, Y., Zhang, H., Zheng, L., Li, S., Hao, H., Yin, M., Cao, Y., Huang, H., 2019. Process Analysis of Anaerobic Fermentation Exposure to Metal Mixtures. Int. J. Env. Res. Pub. He. 16(14), 2458. Viamajala, S., Smith, W.A., Sani, R.K., Apel, W.A., Petersen, J.N., Neal, A.L., Roberto, F.F., Newby, D.T., Peyton, B.M., 2007. Isolation and characterization of Cr(VI) reducing Cellulomonas spp. from subsurface soils: Implications for long-term chromate reduction. Bioresource Technol. 98(3), 612–622. Viti, C., Pace, A., Giovannetti, L., 2003. Characterization of Cr(VI)-resistant bacteria isolated from chromium-contaminated soil by tannery activity. Curr. Microbiol. 46(1), 1–5. Vollmers, J., Wiegand, S., Lenk, F., Kaster, A., 2022. How clear is our current view on microbial dark matter? (Re-)assessing public MAG & SAG datasets with MDMcleaner. Nucleic Acids Res. 50(13), e76. Wang, P.C., Mori, T., Komori, K., Sasatsu, M., Toda, K., Ohtake, H., 1989. Isolation and Characterization of an Enterobacter cloacae Strain That Reduces Hexavalent Chromium under Anaerobic Conditions. Appl Environ Microbiol 55(7), 1665–1669. Wu, Y., Simmons, B.A., Singer, S.W., 2016. MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets. Bioinformatics 32(4), 605–607. Xia, X., Wu, S., Li, N., Wang, D., Zheng, S., Wang, G., 2018. Novel bacterial selenite reductase CsrF responsible for Se(IV) and Cr(VI) reduction that produces nanoparticles in Alishewanella sp. WH16-1. J. Hazard. Mater. 342, 499–509. Xia, X., Wu, S., Zhou, Z., Wang, G., 2021. Microbial Cd(II) and Cr(VI) resistance mechanisms and application in bioremediation. J. Hazard. Mater. 401, 123685. Yan, W., Wang, N., Wei, D., Liang, C., Chen, X., Liu, L., Shi, J., 2021. Bacterial community compositions and nitrogen metabolism function in a cattle farm wastewater treatment plant revealed by Illumina high-throughput sequencing. Environ. Sci. Pollut. R. 28(30), 40895–40907. Yang, W., Hong, W., Huang, Y., Li, S., Li, M., Zhong, H., He, Z., 2022. Exploration on the Cr(VI) resistance mechanism of a novel thermophilic Cr(VI)-reducing bacteriaAnoxybacillus flavithermusABF1 isolated from Tengchong geothermal region, China. Env. Microbiol. Rep. Zhao, KC., Zhang, WJ., Zhentian Liang, Hongyu Zhao, Juanfen Chai, Yuesuo Yang, Tingting Teng, Zhang, DY., 2023. Facilitating New Chromium Reducing Microbes to Enhance Hexavalent Chromium Reduction by In Situ Sonoporation-Mediated Gene Transfer in Soils. Environ. Sci. Technol. 57, 40, 15123–15133. Zhang, B., Wang, Z., Shi, J., Dong, H., 2020. Sulfur-based mixotrophic bio-reduction for efficient removal of chromium (VI) in groundwater. Geochim. Cosmochim. Ac. 268, 296–309. Zhang, J., Zhou, W., Liu, B., He, J., Shen, Q., Zhao, F., 2015. Anaerobic arsenite oxidation by an autotrophic arsenite-oxidizing bacterium from an arsenic-contaminated paddy soil. Environ. Sci. Technol. 49(10), 5956–5964. Zhong, J., Yin, W., Li, Y., Li, P., Wu, J., Jiang, G., Gu, J., Liang, H., 2017. Column study of enhanced Cr(VI) removal and longevity by coupled abiotic and biotic processes using Fe(0) and mixed anaerobic culture. Water Res. 122, 536–544. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8418132","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":602549659,"identity":"096e411b-52df-4fe0-a240-c29a8a9815dc","order_by":0,"name":"Xin Zhang","email":"","orcid":"","institution":"Henan Normal University, Ministry of Education","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhang","suffix":""},{"id":602549660,"identity":"7a875182-f5f1-4824-9d26-22a5e9b2f569","order_by":1,"name":"Zexin Wang","email":"","orcid":"","institution":"Guangdong Institute of Eco-Environmental and Soil Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zexin","middleName":"","lastName":"Wang","suffix":""},{"id":602549661,"identity":"04899478-e7da-4141-89e3-2a092088137f","order_by":2,"name":"Muhammad Usman Ghani","email":"","orcid":"","institution":"Guangdong Institute of Eco-Environmental and Soil Sciences","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Usman","lastName":"Ghani","suffix":""},{"id":602549662,"identity":"37bbd1e6-5fa1-43c0-8199-f4f65fb406f5","order_by":3,"name":"Tianle Kong","email":"","orcid":"","institution":"Guangdong Institute of Eco-Environmental and Soil Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tianle","middleName":"","lastName":"Kong","suffix":""},{"id":602549663,"identity":"706360c8-27e6-4308-bbe6-7d051f7d43a7","order_by":4,"name":"Weimin Sun","email":"","orcid":"","institution":"Guangdong Institute of Eco-Environmental and Soil Sciences","correspondingAuthor":false,"prefix":"","firstName":"Weimin","middleName":"","lastName":"Sun","suffix":""},{"id":602549664,"identity":"cf9dddf9-dbb3-4336-bb0f-c7f0daedadb6","order_by":5,"name":"Xiaoxu Sun","email":"","orcid":"","institution":"Guangdong Institute of Eco-Environmental and Soil Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxu","middleName":"","lastName":"Sun","suffix":""},{"id":602549665,"identity":"12f62691-0446-435b-b6fb-6eea520c6507","order_by":6,"name":"Baoqin Li","email":"","orcid":"","institution":"Guangdong Institute of Eco-Environmental and Soil Sciences","correspondingAuthor":false,"prefix":"","firstName":"Baoqin","middleName":"","lastName":"Li","suffix":""},{"id":602549666,"identity":"1fa2d4d4-f63b-4c98-a7d5-1911f6dc5297","order_by":7,"name":"Zewen Tan","email":"","orcid":"","institution":"Guangdong Institute of Eco-Environmental and Soil Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zewen","middleName":"","lastName":"Tan","suffix":""},{"id":602549667,"identity":"9c5f831b-ada8-4c10-8980-c9751bdc819e","order_by":8,"name":"Geng Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBADOTb2/ocPEipqiNdizM9zhtngwZljxGtJnDkjh03yYQszYaXy7r2HX/PU3GHccCD3WEViAxsDf3t3Al4thmfOpVnzHHvGbHDgXNqNxB0yDBJnzm7Ar2VGjpkxD9thNoODDWY3Es+wMRhI5BLQMv8NUMu/wzwGhxnMChLbmAlrkZfgMX7M23ZYQrKNx4yBKC0GPDlmjHP7Dhvw87AlSyScOcZD0C/y7WeMP7z5dri+Tf7xwY8/Kmrk+Nt7CdhygIFNigdJgAenUrgtDQzMH38QVDYKRsEoGAUjGgAAeQxNPh41GFIAAAAASUVORK5CYII=","orcid":"","institution":"Guangdong Institute of Eco-Environmental and Soil Sciences","correspondingAuthor":true,"prefix":"","firstName":"Geng","middleName":"","lastName":"Yan","suffix":""}],"badges":[],"createdAt":"2025-12-21 15:08:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8418132/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8418132/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104391805,"identity":"56057c23-ea69-4cae-a7b7-53afedb16c1d","added_by":"auto","created_at":"2026-03-11 10:17:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":211725,"visible":true,"origin":"","legend":"\u003cp\u003eConcentrations of Cr(VI) in the live treatment and sterile control inoculated from the XKS sediment (A) and WS paddy soil (B).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8418132/v1/716d6fc6166db9fb44805c42.png"},{"id":104391809,"identity":"3296cd82-0c4e-4ad6-acc2-76250077391d","added_by":"auto","created_at":"2026-03-11 10:17:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":321028,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobial communities at the phylum level in the XKS (A) and WS (B) treatments\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8418132/v1/9b8c3c67438b0d5cdd237f49.png"},{"id":104391808,"identity":"ecc5b0b7-0856-4dcc-bc8b-d570c6602ac1","added_by":"auto","created_at":"2026-03-11 10:17:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":221591,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of domain genera in the XKS (A) and WS (B) treatments.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8418132/v1/fc1fe948338abc83bf3c3e48.png"},{"id":104391806,"identity":"b3cbc4aa-7589-4082-815a-2458dcc22c92","added_by":"auto","created_at":"2026-03-11 10:17:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":335396,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic affiliations of the retrieved genomic bins from XKS (A) and PD (B) samples and the bin quality (i.e., completeness, redundancy, GC content, N50, and size).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8418132/v1/a11bb5c36b7db2c982476cb7.png"},{"id":104391810,"identity":"931c9d38-46fa-441a-b7f5-f400816e0b54","added_by":"auto","created_at":"2026-03-11 10:17:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2771696,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of genes involved in chromate reduction, metal resistance, carbon fixation, sulfur metabolism, and nitrogen metabolism in putative CrRB-associated MAGs.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8418132/v1/08cb27539c0d7acf8d4bfb97.png"},{"id":104405869,"identity":"ed369f23-f302-468b-b327-0e71ed2742ec","added_by":"auto","created_at":"2026-03-11 12:24:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4618925,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8418132/v1/29941cbb-c448-4f53-9743-4718820c0e54.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Deciphering microbial mechanisms for hexavalent chromium reduction in contaminated sediment and paddy soil","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChromium (Cr) is a toxic metal that is extensively distributed in the Earth\u0026rsquo;s crust and has an approximate concentration of 100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Kazakis et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Due to its extensive use in industries, including the manufacture of refractory materials, production of chemical color pigments, and chrome plating, large amounts of Cr are released into the environment, resulting in the severe contamination of soil and water (Rahman and Thomas, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, remediation of Cr contamination has attracted significant attention. Cr occurs in nature in different valence states ranging from -II to +\u0026thinsp;VI. The two dominant species in the environment are Cr(VI) and Cr(III) (Zhong et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Of these two states, Cr(VI) is extremely toxic and exhibits high mobility, whereas Cr(III) is less toxic and tends to form insoluble minerals at neutral pH values (Zhang et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bishop et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, the reduction of Cr(VI) to Cr(III) contributes to the remediation of Cr-contaminated sites and rehabilitation of environments affected by Cr contamination.\u003c/p\u003e \u003cp\u003eMicrobially mediated Cr(VI) reduction is an efficient remediation strategy that has attracted the interest of the scientific community due to its cost-effectiveness and environmental sustainability (Le et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Min et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In microbial Cr(VI) reduction, electron donors play an important role in the biogeochemical process, and various electron donors have been used, such as organic matter, CH\u003csub\u003e4\u003c/sub\u003e, Fe(0), S(0), and H\u003csub\u003e2\u003c/sub\u003e (Lara et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; He et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lai et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Long et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Shi et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, several Cr(VI)-reducing bacteria (CrRBs) have been isolated from various habitats, including \u003cem\u003eEnterobacter cloacae\u003c/em\u003e HO1 (Wang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), \u003cem\u003eCellulomonas tarbata\u003c/em\u003e (Viti et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), \u003cem\u003ePseudomonas\u003c/em\u003e sp. G1DM21 (Desai et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), \u003cem\u003eBacillus cereus\u003c/em\u003e (He et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), \u003cem\u003eLysinibacillus\u003c/em\u003e sp. (Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and \u003cem\u003eLactococcus lactis\u003c/em\u003e AM99 (Akhzari et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Most of these bacteria are heterogeneous because organics can support high microbial activity, making heterotrophic Cr(VI) reduction a promising remediation technology. However, the potential use of Cr(VI)-reducing bacteria, particularly indigenous microbial communities, in the remediation of Cr(VI)-contaminated environments may be underestimated because of the inherent limitations of culture-dependent analytical techniques.\u003c/p\u003e \u003cp\u003eMost studies have focused on identifying CrRBs and evaluating their chromate transport capability. For instance, \u003cem\u003eAnoxybacillus flavithermus\u003c/em\u003e ABF1 was isolated as an alkaline Cr(VI)-reducing bacterium from a geothermal region, and was able to reduce 93.71% of Cr(VI) at an initial concentration of 20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 72 hours (Yang et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Members of the \u003cem\u003eComamonadaceae\u003c/em\u003e family have been identified in methane- and oxygen-based membrane biofilm reactor with the potential for chromate reduction, which could reduce Cr(VI) to Cr(III) at a rate of 89% (Long et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Zhang et al. (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) proposed a mechanism for the metabolic pathway of Cr(VI)-reducing bacteria in a sulfur-based mixotrophic system by determining the abundance of the \u003cem\u003echrA\u003c/em\u003e genes and the compounds involved in Cr(VI) reduction (Zhang et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the reduction mechanisms of most Cr(VI)-reducing bacteria are not clearly understood because most of them have not been isolated, resulting in a lack of molecular biological evidence. Therefore, a better understanding of the metabolic mechanisms of Cr(VI)-reducing bacteria is required.\u003c/p\u003e \u003cp\u003eIn this study, anaerobic cultures were established to enrich indigenous Cr(VI)-reducing bacteria using long-term Cr(VI)-contaminated sediments and paddy soils as inocula. A metagenomic approach was used to reconstruct the metagenome-assembled genomes (MAGs) of CrRBs from these complex microbial communities and predict the Cr(VI) reduction pathway. The aim of the current study was to investigate (i) which microorganisms are responsible for Cr(VI)-reducing bacteria in Cr(VI)-contaminated sediments and paddy soils and (ii) the metabolic mechanisms of CrRBs. These findings provide new insights into the diversity and metabolic potential of CrRBs for Cr(VI) reduction, which may contribute to the remediation of Cr(VI) in contaminated environments.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sample collection and characterization\u003c/h2\u003e \u003cp\u003eSamples were collected from river sediments at depth of 0\u0026ndash;20 cm in Xikuangshan, Hunan, China (27\u0026deg;44\u0026prime; N, 111\u0026deg;28\u0026prime; E, designated as XKS), and from flooded rice fields in Wanshan, Guizhou, China (27\u0026deg;31\u0026prime; N, 109\u0026deg;12\u0026prime; E, designated as WS), both located near the mining area. The collected samples were immediately transported to the laboratory on ice and stored at -20 ℃ for chemical analysis and microcosm incubation. To determine the Cr content, the samples were freeze-dried, digested with an HCl: HNO\u003csub\u003e3\u003c/sub\u003e solution (3:1, v/v), and measured by ICP-OES (Vista MPX, Varian). The samples of XKS and WS contained 237.3 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 136.8 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Cr, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Anaerobic enrichment cultures\u003c/h2\u003e \u003cp\u003eAnaerobic enrichment cultures were established using the collected samples to detect potential Cr-reducing bacteria. Approximately 2 g of the sample was added to 100 mL of mineral salt medium (MSM; 7.9 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 1.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NH\u003csub\u003e4\u003c/sub\u003eCl, 0.1 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 10 mL L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e vitamin solution, and 5 mL L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e trace element solution) as inoculum (Zhang et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The bottles were then purged with N\u003csub\u003e2\u003c/sub\u003e for 30 min, capped with butyl rubber septa and aluminum caps, and inoculated for three days at room temperature to degrade soluble organics and indigenous Cr in the original inoculum. Cr(VI) was added to the cultures at a final concentration of 20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 6 mM sodium acetate was used as the electron donor. In addition, abiotic controls were prepared using autoclaved samples to demonstrate that the biological activity mediates this process. All cultures were prepared in triplicates at each sampling time point and incubated at 30 \u003csup\u003eo\u003c/sup\u003eC. During incubation, the Cr(VI) concentration was monitored by taking 1 mL of supernatant from each culture for colorimetric measurement according to the established protocol (He et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). After the complete removal of Cr(VI), the cultures were spiked again with 20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Cr(VI) (which was considered as one cycle), and duplicate cultures were destructively sampled for microbial community analysis. Three samples were analyzed for each case.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 DNA extraction\u003c/h2\u003e \u003cp\u003eCultures collected from each enrichment cycle were centrifuged at 7,000 rpm for 10 min. Total genomic DNA was extracted from the cultures and raw samples using the DNeasy PowerSoil DNA Kit (QIAGEN, Germany) according to the manufacturer\u0026rsquo;s protocol. DNA was not extracted from the sterile controls in this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Illumina MiSeq sequencing of 16S rRNA gene amplicons\u003c/h2\u003e \u003cp\u003eTo characterize the prokaryotic communities, the hypervariable region V4 of the 16S rRNA gene was amplified using extracted DNA as a template. The forward primer was 515F (5\u0026rsquo;-GTGYCAGCMGCCGCGGTAA-3\u0026rsquo;) and the reverse primer was 806R (5\u0026rsquo;-GGACTACNVGGGTWTCTAAT-3\u0026rsquo;) (Klindworth et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The amplification products were sequenced on an Illumina MiSeq platform at Personal Biotechnology (Shanghai, China). The raw amplicon sequencing data were analyzed using the QIIME2-202002 toolkit (Bolyen et al., 2019). Briefly, raw reads were merged, trimmed, and filtered. The filtered reads were clustered into amplicon sequence variants (ASV) using DADA2 (Callahan et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Representative sequences were assigned to the SILVA 132 database (Quast et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Shotgun metagenomic sequencing and analysis\u003c/h2\u003e \u003cp\u003eIn the third cycle, DNA from triplicate cultures was combined to create a composite DNA sample because the amount of DNA extracted from each culture was insufficient for shotgun metagenomic sequencing. The DNA samples were then sequenced on an Illumina HiSeq 4000 platform (PersonalBio, Shanghai, China). Approximately 33,934,255 and 33,985,822 raw reads were generated from the XKS and WS metagenomes, respectively. All raw reads were trimmed using Trimmomatic 0.39 (Bolger et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) with the following arguments: \u0026ldquo;sliding window: 4:15, leading: 3, trailing: 3, and minilen: 80\u0026rdquo;. Overlapping read pairs were identified and merged using FLASH with a minimum overlap of 15 bp and a maximum overlap of 100 bp. Clean datasets were assembled de novo into contigs using MEGAHIT (Li et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) (k\u0026thinsp;=\u0026thinsp;21\u0026ndash;141, step\u0026thinsp;=\u0026thinsp;10). Metagenome-assembled genomes (MAGs) were retrieved from the assembled contigs by metagenome binning using MaxBin2 (Wu et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), MetaBAT2 (Kang et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and CONCOCT (Alneberg et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The obtained MAGs were refined and their quality was improved using DAS (Sieber et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The MAGs were then filtered using an MDMcleaner (Vollmers et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and their quality was assessed using CheckM (Parks et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Only MAGs with a completion rate of \u0026gt;\u0026thinsp;60% and contamination rate of \u0026lt;\u0026thinsp;10% were retained and subjected to taxonomic annotation with GTDB-TK (Chaumeil et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The functional genes of the MAGs were annotated using KofamKOALA (Aramaki et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with default parameters. The phylogeny of MAGs genomes was generated using MEGA (Tamura et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Data availability\u003c/h2\u003e \u003cp\u003eNucleic acid reads based on the 16S rRNA gene and metagenomic sequencing were submitted to the NCBI GenBank database under project number PRJNA847997.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Microbial reduction of Cr(VI)\u003c/h2\u003e \u003cp\u003eAnaerobic enrichment cultures were established using two Cr-contaminated samples as the inocula. Cr(VI) was added for three cycles as the sole electron acceptor to enrich the Cr(VI)-reducing bacteria. The concentration of aqueous Cr(VI) was monitored during the incubation period \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The Cr(VI) concentration constantly decreased in the two live treatments inoculated with the XKS sediment and WS paddy soil. In contrast, the Cr(VI) concentration in the sterile control initially decreased slightly and then reached a metastable state. These observations suggest that the reduction of Cr(VI) was driven by microorganisms. Additionally, the Cr(VI) reduction rates were significantly different between the two live treatments. Approximately 20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Cr(VI) was reduced to 0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e within 6 days in the XKS treatment, while 12 days were required for the complete reduction of Cr(VI) in the WS treatment, suggesting a higher reduction potential of Cr(VI) in the XKS treatment than in the WS treatment. The different microbial communities in the two treatments may have contributed to this outcome, or the XKS treatment may have resulted in a higher abundance of potential Cr(VI)-reducing bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Enrichment of Cr(VI) reducing bacteria (CrRB)\u003c/h2\u003e \u003cp\u003eSimilar to the original inoculum, the microbial communities in both cultures were characterized at the end of each cycle. Different microbial taxa were enriched in XKS and WS cultures, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. At the phylum level, the relative abundance of \u003cem\u003eBacteroidota\u003c/em\u003e in the XKS cultures gradually increased during the incubation period and reached the level of the dominant population (36%) after three cycles. At the same time, the relative abundance of \u003cem\u003eDesulfobacterota\u003c/em\u003e in the XKS cultures increased from 2% to 16%. During incubation, the relative abundances of \u003cem\u003eActinobacteriota\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e in the WS cultures were 7% and 11%, respectively, whereas those in the original inoculum were 42% and 36%, respectively. Additionally, \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e were the dominant bacteria in the XKS and WS cultures, accounting for 64% and 65% of the total abundance, respectively, while their abundance decreased to 18% and 13%, respectively, at the end of incubation. These findings suggest that distinct microbial communities mediate Cr(VI) reduction and reveal that Cr(VI) exerts a strong selective pressure on different microorganisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the genus level, uncultured \u003cem\u003eRikenellaceae\u003c/em\u003e became the most abundant taxon in the XKS enrichment cultures, with a relative abundance of 21% in the third cycle compared to 0.4% in the original inoculum, followed by \u003cem\u003eCitrifermentans\u003c/em\u003e with a relative abundance of 7% \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. After three cycles of incubation, bacteria associated with \u003cem\u003eCellulomonas\u003c/em\u003e and \u003cem\u003eEnterobacter\u003c/em\u003e gradually dominated, with relative abundances of 36% and 28%, respectively, in the WS cultures. In addition, the relative abundances of other genera, such as \u003cem\u003eThiobacillus\u003c/em\u003e, \u003cem\u003eBrevundimonas\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e, and \u003cem\u003eDesulfitobacterium\u003c/em\u003e, decreased with incubation time. These findings suggest that uncultured \u003cem\u003eRikenellaceae\u003c/em\u003e, \u003cem\u003eCitrifermentans\u003c/em\u003e, \u003cem\u003eCellulomonas\u003c/em\u003e, and \u003cem\u003eEnterobacter\u003c/em\u003e may be responsible for Cr(VI) reduction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Metagenomic analysis of CrRBs\u003c/h2\u003e \u003cp\u003eMetagenomic binning was performed to retrieve genomic information on the proposed CrRBs and further elucidate their metabolic potential. A total of 21 metagenomic-assembled genomes (MAGs) with \u0026gt;\u0026thinsp;60% completeness and \u0026lt;\u0026thinsp;10% contamination were obtained from the two metagenomes. These MAGs were categorized into seven different bacterial phyla: \u003cem\u003eHalobacteriota\u003c/em\u003e, \u003cem\u003eAcidobacteriota\u003c/em\u003e, \u003cem\u003eChloroflexota\u003c/em\u003e, \u003cem\u003eBacteroidota\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eDesulfobacterota\u003c/em\u003e, and \u003cem\u003eActinobacteriota\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Among the recovered MAGs, seven were taxonomically linked to putative CrRB, including \u003cem\u003eRikenellaceae_uncultured\u003c/em\u003e (bin.5 and bin.8), \u003cem\u003eCitrifermentans\u003c/em\u003e (bin.9 and bin.11), \u003cem\u003eCellulomonas\u003c/em\u003e (bin.4 and bin.5) and \u003cem\u003eEnterobacter\u003c/em\u003e (bin.1), which were selected for further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFunctional genes of MAGs associated with CrRBs were identified \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Key genes involved in chromate reduction (\u003cem\u003echrR\u003c/em\u003e) and chromate transport (\u003cem\u003echrA\u003c/em\u003e) (He et al., 2018; Lu et al., 2020) were detected in all putative CrRB-associated MAGs except for \u003cem\u003eCellulomonas\u003c/em\u003e-associated MAGs. In addition, other genes potentially involved in Cr(VI) reduction have been examined in these MAGs, including \u003cem\u003essuE\u003c/em\u003e, \u003cem\u003eazoR\u003c/em\u003e, \u003cem\u003erutE\u003c/em\u003e, \u003cem\u003enfsA\u003c/em\u003e, \u003cem\u003eribF\u003c/em\u003e, \u003cem\u003enemA\u003c/em\u003e, \u003cem\u003enrfA\u003c/em\u003e, and \u003cem\u003emodA\u003c/em\u003e (Xia et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xia et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These observations suggested that these bacteria have the potential to reduce Cr(VI). In addition, putative CrRB-associated MAGs harbored diverse genes related to metal resistance, such as \u003cem\u003eczcABCD\u003c/em\u003e, arsC, arsR, cusB, and znuABC, suggesting their genetic potential to adapt to metal-contaminated habitats. All seven CrRB-associated MAGs harbored genes encoding for six major carbon fixation pathways, indicating their ability to grow with acetic acid. In addition, some genes involved in nitrogen and sulfur metabolism were detected in the CrRB-associated MAGs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMicrobial-driven Cr(VI) reduction has important environmental implications for the remediation of Cr-contaminated environments and has been widely studied in natural habitats, such as soils (Hou et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Fu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and groundwater (Shi et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, the diversity and metabolic potential of Cr(VI)-reducing microbial communities in sediments and paddy soils were investigated using anaerobic enrichment cultures and metagenomics.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Potential Cr(VI)-reducing bacteria\u003c/h2\u003e \u003cp\u003eBiological Cr(VI) reduction has been observed in the cultures inoculated with Cr-contaminated sediment and paddy soil from two sites whose aqueous Cr(VI) reduced in 6 and 12 days, respectively. Four bacteria, \u003cem\u003eRikenellaceae\u003c/em\u003e, \u003cem\u003eCitrifermentans\u003c/em\u003e, \u003cem\u003eCellulomonas\u003c/em\u003e, and \u003cem\u003eEnterobacter\u003c/em\u003e, were identified as putative CrRBs based on their relative abundances in the cultures, which increased significantly with incubation time. After incubation, the dominant taxa in the cultures among these CrRBs were \u003cem\u003eRikenellaceae\u003c/em\u003e, \u003cem\u003eCellulomonas\u003c/em\u003e, and \u003cem\u003eEnterobacter\u003c/em\u003e, with relative abundances of more than 20%.\u003c/p\u003e \u003cp\u003eAnaerobic fermentation bacteria associated with the \u003cem\u003eRikenellaceae\u003c/em\u003e family have been found in anaerobic environments including swamps (Su et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and anaerobic activated sludge (Yan et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Some studies have shown that members of the \u003cem\u003eRikenellaceae\u003c/em\u003e are enriched during the decomposition and digestion of organic matter (Tian et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), suggesting that they may have the ability to utilize acetate. It has been reported that \u003cem\u003eRikenellaceae\u003c/em\u003e are potentially syntrophic bacteria that can directly transfer electron with methanogens through iron oxide minerals (Lee et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, slight enrichment of \u003cem\u003eRikenellaceae\u003c/em\u003e was observed in the reduction of Cr(VI) and nitrate driven by the microbiome and iron oxide minerals (Hu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), suggesting that \u003cem\u003eRikenellaceae\u003c/em\u003e may have the potential to reduce or tolerate Cr(VI). However, the ability of \u003cem\u003eRikenellaceae\u003c/em\u003e spp. to reduce Cr(VI) has not yet been reported.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEnterobacter\u003c/em\u003e spp. have been previously identified as Cr-reducing bacteria. \u003cem\u003eEnterobacter cloacae\u003c/em\u003e HO1 was the first strain to have the ability to reduce Cr(VI) (Wang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Various members of \u003cem\u003eEnterobacter\u003c/em\u003e have been isolated, including \u003cem\u003eEnterobacter aerogenes\u003c/em\u003e T2 (Panda and Sarkar, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), \u003cem\u003eEnterobacter cloacae\u003c/em\u003e B2-DHA (Rahman et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and \u003cem\u003eEnterobacter\u003c/em\u003e sp. SL (Sun et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eEnterobacter cloacae\u003c/em\u003e B2-DHA can resist 1000 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Cr(VI) (Rahman et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and \u003cem\u003eEnterobacter\u003c/em\u003e sp. DU17 reduced Cr(VI) by 100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e over 16 h using glucose as an electron donor (Rahman and Singh, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), suggesting its strong ability to reduce Cr(VI). In addition, Cr(VI) reduction was effectively observed when \u003cem\u003eEnterobacter cloacae\u003c/em\u003e HO1 was enriched with acetate compared to glucose, citrate, or lactate (Komori et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), suggesting that acetate is an efficient electron donor for \u003cem\u003eEnterobacter cloacae\u003c/em\u003e HO1 to reduce Cr(VI). In agreement with our current study, \u003cem\u003eEnterobacter\u003c/em\u003e dominated with increasing incubation time when acetate was used as the electron donor.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCellulomonas\u003c/em\u003e spp. are known to reduce Cr(VI) and some of their members have been isolated from various habitats (Brookshier et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Viamajala et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003eb). For example, \u003cem\u003eCellulomonas tarbata\u003c/em\u003e isolated from Cr-contaminated soil was resistant to Cr(VI) concentrations up to 12 mM (Viti et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In another study, \u003cem\u003eCellulomonas\u003c/em\u003e WS01 reduced 0.2 mM of Cr(VI) in 70 hours (Sani et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). These results indicated that bacteria associated with \u003cem\u003eCellulomonas\u003c/em\u003e can reduce Cr(VI). Additionally, \u003cem\u003eCellulomonas\u003c/em\u003e ES6 was isolated from Cr-contaminated sediment reduced anaerobically with Cr(VI) using acetate as an electron donor (Viamajala et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003eb), similar to the conditions used in this study.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCitrifermentans\u003c/em\u003e are a rarely studied group of anaerobic bacteria and only three strains have been isolated from this genus: \u003cem\u003eCitrifermentans bemidjiense\u003c/em\u003e (Nevin et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), \u003cem\u003eCitrifermentans bremense\u003c/em\u003e, and \u003cem\u003eCitrifermentans pelophilum\u003c/em\u003e (Straub and Buchholz-Cleven, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). These three bacteria have been previously identified as Fe(III)-reducing bacteria (Straub and Buchholz-Cleven, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Aklujkar et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Neal et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), indicating their potential for metal reduction. Although some members of the \u003cem\u003eGeobacteraceae\u003c/em\u003e family have been reported to reduce Cr(VI) (Shi et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; He et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), identification of the genus \u003cem\u003eCitrifermentans\u003c/em\u003e has not yet been reported.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Metabolic Potentials of CrRB-Associated MAGs\u003c/h2\u003e \u003cp\u003eThe genetic traits responsible for Cr(VI) reduction are of particular significance as they may represent some of the most significant characteristics for the bioremediation of Cr. Metagenomic binning was employed to retrieve bins associated with putative Cr(VI)-reducing bacteria, thereby enabling the exploration of the underlying mechanisms involved in Cr(VI) reduction. Various gene families hypothesized to be involved in Cr(VI) reduction were selected to examine the presence of these genes within the MAGs linked to putative Cr(VI)-reducing bacteria. C\u003cem\u003ehrR\u003c/em\u003e, which belongs to the flavodoxin superfamily, is a chromate reductase (Eswaramoorthy et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A reductase in the NADPH-dependent flavoprotein family, ChrA, also plays a key role in Cr(VI) reduction and resistance (Ramirez-Diaz et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). During reduction, ChrR transfers NADH electrons to Cr(VI) and directly reduces Cr(VI), whereas ChrA serves as a chromate transporter that exports Cr(III) into the extracellular environment. In this study, \u003cem\u003echrR\u003c/em\u003e was detected in MAGs associated with \u003cem\u003eCitrifermentans\u003c/em\u003e and \u003cem\u003eEnterobacter\u003c/em\u003e, whereas \u003cem\u003echrA\u003c/em\u003e was detected in MAGs from \u003cem\u003eRikenellaceae\u003c/em\u003e, \u003cem\u003eCitrifermentans\u003c/em\u003e, and \u003cem\u003eEnterobacter\u003c/em\u003e, suggesting that either or both enzymes may be used to reduce Cr(VI). Neither \u003cem\u003echrR\u003c/em\u003e nor \u003cem\u003echrA\u003c/em\u003e were detected in \u003cem\u003eCellulomonas\u003c/em\u003e-associated MAGs, which may be attributed to incomplete metagenomic binning that did not provide the whole genomes of these bacteria. The genomes of the \u003cem\u003eCellulomonas\u003c/em\u003e sp. strains K38 and B12 were annotated using chromate reductases (ChrRs) (Brookshier et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to \u003cem\u003echrR\u003c/em\u003e and \u003cem\u003echrA\u003c/em\u003e, Cr(VI) reduction has been observed to be mediated by flavoprotein reductase (RibF) in Cr(VI)-reducing \u003cem\u003eSerratia\u003c/em\u003e sp. S2 (Dong et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This gene was observed in all the putative CrRB-associated MAGs. Some genes have been reported to be responsible for Cr(VI) reduction in various bacteria including \u003cem\u003enrfA\u003c/em\u003e, \u003cem\u003essuE\u003c/em\u003e, \u003cem\u003erutE\u003c/em\u003e, \u003cem\u003eazoR\u003c/em\u003e, \u003cem\u003enemA\u003c/em\u003e, and \u003cem\u003enfsA\u003c/em\u003e (Yang et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bhattacharya et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Xia et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These genes were detected in putative CrRB-associated MAGs, indicating that putative CrRBs may use various pathways for Cr(VI) reduction. These bacteria were further verified as putative Cr(VI) reducers by identifying potential functional genes for Cr(VI) reduction.\u003c/p\u003e \u003cp\u003eIn addition, putative CrRB-associated MAGs harbored several genes involved in resistance to different metals. For instance, most MAGs harbor the \u003cem\u003earsR\u003c/em\u003e and \u003cem\u003earsC\u003c/em\u003e genes, both of which are involved in As and Sb resistance and their detoxification pathways (Ji et al., 1992). Genes encoding Cd, Zn, Cu, and Mn exporters were identified in all MAGs, suggesting that CrRBs can resist or transform these metals, because the samples used in this study were contaminated with various metals.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, enrichment incubation and metagenomics were combined to enrich CrRBs and to elucidate the metabolic pathways involved in Cr(VI) reduction. After three enrichment cycles, \u003cem\u003eCellulomonas\u003c/em\u003e, \u003cem\u003eEnterobacter\u003c/em\u003e, \u003cem\u003eRikenellaceae\u003c/em\u003e, and \u003cem\u003eCitrifermentans\u003c/em\u003e dominated Cr-contaminated sediments and paddy soils, with abundances of 36, 28, 21, and 7%, respectively, indicating that these were putative CrRBs. Among these, \u003cem\u003eRikenellaceae\u003c/em\u003e and \u003cem\u003eCitrifermentans\u003c/em\u003e are novel chromium-reducing bacteria. The reconstructed genomes of the four putative CrRBs encoded key genes (\u003cem\u003echrA\u003c/em\u003e and \u003cem\u003echrR\u003c/em\u003e) for chromate reduction, indicating their ability to reduce chromate. This study expands the known diversity of Cr(VI)-reducing bacteria and improves our understanding of their metabolism.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors \u0026nbsp;contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXin Zhang\u003c/strong\u003e: Writing - Original Draft, Investigation, Data curation,Formal analysis,Methodology. \u003cstrong\u003eZexin Wang\u003c/strong\u003e: Data curation,Investigation, Writing - Original Draft,Writing - Review \u0026amp; Editing. \u003cstrong\u003eMuhammad Usman Ghani\u003c/strong\u003e:Investigation,Data curation,Writing - Review \u0026amp; Editing. \u003cstrong\u003eTianle Kong\u003c/strong\u003e:Investigation,Methodology,Validation.\u003cstrong\u003eWeimin Sun\u003c/strong\u003e: Formal analysis,Funding acquisition, Writing - Review \u0026amp; Editing.\u003cstrong\u003eXiaoxu Sun\u003c/strong\u003e:Funding acquisition, Writing - Review \u0026amp; Editing. \u003cstrong\u003eBaoqin Li\u003c/strong\u003e: Visualization,Writing - Review \u0026amp; Editing. \u003cstrong\u003eZewen Tan\u003c/strong\u003e: Data curation, Writing - Review \u0026amp; Editing. \u003cstrong\u003eGeng Yan\u0026nbsp;\u003c/strong\u003e:Conceptualization,Funding acquisition,Supervision,Writing - Review \u0026amp; Editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (Grant No. 2023YFC3706903), the National Natural Science Foundation of China (Grant Nos. U21A2035, 42321005, 42307354 and 42277247), Guangdong Basic and Applied Basic Research Foundation (Grant Nos. 2025B1515020024, 2023B1515040007, 2022B1515120033, 2024A1515011436 and 2024A1515011044), GDAS\u0026apos; Project of Science and Technology Development (Grant Nos. 2024GDASQNRC-0108 and 2023GDASZH-2023010104-1), China Postdoctoral Science Foundation funded Project (Grant No. 2023M740763), and Guangdong Foundation for Program of Science and Technology Research (Grant No. 2023B1212060044).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets obtained during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics Approval\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e This manuscript is approved by all authors for publication in Water, Air, \u0026amp; Soil Pollution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkhzari, F., Naseri, T., Mousavi, SM., Khosravi-Darani, K., 2024. A sustainable solution for alleviating hexavalent chromium from water streams using \u003cem\u003eLactococcus lactis\u003c/em\u003e AM99 as a novel Cr(VI)-reducing bacterium. J Environ Manage. 353, 120190.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAklujkar, M., Young, N.D., Holmes, D., Chavan, M., Risso, C., Kiss, H.E., Han, C.S., Land, M.L., Lovley, D.R., 2010. The genome of Geobacter bemidjiensis, exemplar for the subsurface clade of Geobacter species that predominate in Fe(III)-reducing subsurface environments. BMC Genomics 11, 490.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlneberg, J., Bjarnason, B.S., de Bruijn, I., Schirmer, M., Quick, J., Ijaz, U.Z., Lahti, L., Loman, N.J., Andersson, A.F., Quince, C., 2014. Binning metagenomic contigs by coverage and composition. Nat. Methods 11(11), 1144\u0026ndash;1146.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAramaki, T., Blanc-Mathieu, R., Endo, H., Ohkubo, K., Kanehisa, M., Goto, S., Ogata, H., 2020. KofamKOALA: KEGG Ortholog assignment based on profile HMM and adaptive score threshold. Bioinformatics 36(7), 2251\u0026ndash;2252.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhattacharya, P., Barnebey, A., Zemla, M., Goodwin, L., Auer, M., Yannone, S.M., 2015. Complete genome sequence of the chromate-reducing bacterium Thermoanaerobacter thermohydrosulfuricus strain BSB-33. Stand. Genomic Sci. 10(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBishop, M.E., Dong, H., Glasser, P., Briggs, B.R., Pentrak, M., Stucki, J.W., Boyanov, M.I., Kemner, K.M., Kovarik, L., 2019. Reactivity of redox cycled Fe-bearing subsurface sediments towards hexavalent chromium reduction. Geochim. Cosmochim. Ac. 252, 88\u0026ndash;106.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15), 2114\u0026ndash;2120.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolyen, E., Rideout, J.R., Dillon, M.R., Bokulich, N.A., Abnet, C.C., Al-Ghalith, G.A., Alexander, H., Alm, E.J., Arumugam, M., Asnicar, F., Bai, Y., Bisanz, J.E., Bittinger, K., Brejnrod, A., Brislawn, C.J., Brown, C.T., Callahan, B.J., Caraballo-Rodr\u0026iacute;guez, A.M., Chase, J., Cope, E.K., Da Silva, R., Diener, C., Dorrestein, P.C., Douglas, G.M., Durall, D.M., Duvallet, C., Edwardson, C.F., Ernst, M., Estaki, M., Fouquier, J., Gauglitz, J.M., Gibbons, S.M., Gibson, D.L., Gonzalez, A., Gorlick, K., Guo, J., Hillmann, B., Holmes, S., Holste, H., Huttenhower, C., Huttley, G.A., Janssen, S., Jarmusch, A.K., Jiang, L., Kaehler, B.D., Kang, K.B., Keefe, C.R., Keim, P., Kelley, S.T., Knights, D., Koester, I., Kosciolek, T., Kreps, J., Langille, M.G.I., Lee, J., Ley, R., Liu, Y., Loftfield, E., Lozupone, C., Maher, M., Marotz, C., Martin, B.D., McDonald, D., McIver, L.J., Melnik, A.V., Metcalf, J.L., Morgan, S.C., Morton, J.T., Naimey, A.T., Navas-Molina, J.A., Nothias, L.F., Orchanian, S.B., Pearson, T., Peoples, S.L., Petras, D., Preuss, M.L., Pruesse, E., Rasmussen, L.B., Rivers, A., Robeson, M.S., Rosenthal, P., Segata, N., Shaffer, M., Shiffer, A., Sinha, R., Song, S.J., Spear, J.R., Swafford, A.D., Thompson, L.R., Torres, P.J., Trinh, P., Tripathi, A., Turnbaugh, P.J., Ul-Hasan, S., van der Hooft, J.J.J., Vargas, F., V\u0026aacute;zquez-Baeza, Y., Vogtmann, E., von Hippel, M., Walters, W., Wan, Y., Wang, M., Warren, J., Weber, K.C., Williamson, C.H.D., Willis, A.D., Xu, Z.Z., Zaneveld, J.R., Zhang, Y., Zhu, Q., Knight, R., Caporaso, J.G., 2019. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37(9), 1091.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrookshier, A.M., Santo, D.J., Kourtev, P.S., Learman, D.R., 2018. Draft Genome Sequences of Two Bacillus sp. Strains and Four Cellulomonas sp. Strains Isolated from Heavy-Metal-Contaminated Soil. Microbiol Resour Announc 7(11).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCallahan, B.J., McMurdie, P.J., Rosen, M.J., Han, A.W., Johnson, A.J.A., Holmes, S.P., 2016. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13(7), 581\u0026ndash;583.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChai, L., Ding, C., Tang, C., Yang, W., Yang, Z., Wang, Y., Liao, Q., Li, J., 2018. Discerning three novel chromate reduce and transport genes of highly efficient Pannonibacter phragmitetus BB: From genome to gene and protein. Ecotox. Environ. Safe. 162, 139\u0026ndash;146.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaumeil, P., Mussig, A.J., Hugenholtz, P., Parks, D.H., 2019. GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, J., Li, X., Gan, L., Jiang, G., Zhang, R., Xu, Z., Tian, Y., 2021. Mechanism of Cr(VI) reduction by Lysinibacillus sp. HST-98, a newly isolated Cr (VI)-reducing strain. Environ Sci Pollut Res Int 28(46), 66121\u0026ndash;66132.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, J., Li, X., Gan, L., Jiang, G., Zhang, R., Xu, Z., Tian, Y., 2021. Mechanism of Cr(VI) reduction by Lysinibacillus sp. HST-98, a newly isolated Cr (VI)-reducing strain. Environ. Sci. Pollut. R. 28(46), 66121\u0026ndash;66132.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, J., Yang, Y., Ke, Y., Chen, X., Jiang, X., Chen, C., Xie, S., 2022. Anaerobic sulfamethoxazole-degrading bacterial consortia in antibiotic‐contaminated wetland sediments identified byDNA ‐stable isotope probing and metagenomics analysis. Environ. Microbiol. 24(8), 3751\u0026ndash;3763.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDesai, C., Jain, K., Madamwar, D., 2008. Hexavalent chromate reductase activity in cytosolic fractions of Pseudomonas sp. G1DM21 isolated from Cr(VI) contaminated industrial landfill. Process Biochem. 43(7), 713\u0026ndash;721.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong, L., Zhou, S., He, Y., Jia, Y., Bai, Q., Deng, P., Gao, J., Li, Y., Xiao, H., 2018. Analysis of the Genome and Chromium Metabolism-Related Genes of Serratia sp. S2. Appl. Biochem. Biotech. 185(1), 140\u0026ndash;152.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEswaramoorthy, S., Poulain, S., Hienerwadel, R., Bremond, N., Sylvester, M.D., Zhang, Y.B., Berthomieu, C., Van Der Lelie, D., Matin, A., 2012. Crystal structure of ChrR\u0026ndash;a quinone reductase with the capacity to reduce chromate. PLoS One 7(4), e36017.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu, L., Feng, A., Xiao, J., Wu, Q., Ye, Q., Peng, S., 2021. Remediation of soil contaminated with high levels of hexavalent chromium by combined chemical-microbial reduction and stabilization. J. Hazard. Mater. 403, 123847.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe, C., Zhang, B., Yan, W., Ding, D., Guo, J., 2020. Enhanced Microbial Chromate Reduction Using Hydrogen and Methane as Joint Electron Donors. J. Hazard. Mater. 395, 122684.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe, M., Li, X., Guo, L., Miller, S.J., Rensing, C., Wang, G., 2010. Characterization and genomic analysis of chromate resistant and reducing Bacillus cereus strain SJ1. BMC Microbiol. 10, 221.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe, Y., Gong, Y., Su, Y., Zhang, Y., Zhou, X., 2019. Bioremediation of Cr (VI) contaminated groundwater by Geobacter sulfurreducens: Environmental factors and electron transfer flow studies. Chemosphere 221, 793\u0026ndash;801.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHou, S., Wu, B., Luo, Y., Li, Y., Ma, H., Peng, D., Xu, H., 2020. Impacts of a novel strain QY-1 allied with chromium immobilizing materials on chromium availability and soil biochemical properties. J. Hazard. Mater. 382, 121093.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu, Y., Liu, T., Chen, N., Feng, C., 2021. Iron oxide minerals promote simultaneous bio-reduction of Cr(VI) and nitrate: Implications for understanding natural attenuation. Sci. Total Environ. 786, 147396.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang, D.D., Froula, J., Egan, R., Wang, Z., 2015. MetaBAT, an efficient tool for accurately reconstructing single genomes from complex microbial communities. PeerJ 3, e1165.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKazakis, N., Kantiranis, N., Voudouris, K.S., Mitrakas, M., Kaprara, E., Pavlou, A., 2015. Geogenic Cr oxidation on the surface of mafic minerals and the hydrogeological conditions influencing hexavalent chromium concentrations in groundwater. Sci. Total Environ. 514, 224\u0026ndash;238.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlindworth, A., Pruesse, E., Schweer, T., Peplies, J., Quast, C., Horn, M., Gl\u0026ouml;ckner, F.O., 2013. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 41(1), e1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKOMORI, K., WANG, P., TODA, K., OHTAKE, H., 1989. Factors affecting chromate reduction in Enterobacter cloacae strain HO1. Appl. Microbiol. Biot. 31(5\u0026ndash;6), 567\u0026ndash;570.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLai, C., Zhong, L., Zhang, Y., Chen, J., Wen, L., Shi, L., Sun, Y., Ma, F., Rittmann, B.E., Zhou, C., Tang, Y., Zheng, P., Zhao, H., 2016. Bioreduction of Chromate in a Methane-Based Membrane Biofilm Reactor. Environ. Sci. Technol. 50(11), 5832\u0026ndash;5839.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLara, P., Morett, E., Ju\u0026aacute;rez, K., 2017. Acetate biostimulation as an effective treatment for cleaning up alkaline soil highly contaminated with Cr(VI). Environ. Sci. Pollut. R. 24(33), 25513\u0026ndash;25521.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, J., Koo, T., Yulisa, A., Hwang, S., 2019. Magnetite as an enhancer in methanogenic degradation of volatile fatty acids under ammonia-stressed condition. J. Environ. Manage. 241, 418\u0026ndash;426.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYilin Le, Xing He, Mengnan Liu, Xue Liu, Shidong Zhou, Rongrong Xie, Yu Fu, Huilei Wang, Jianzhong Sun., 2023. Light-driven sustainable enhancement of Cr(VI) reduction via the combination of Cr(VI)-reducing bacteria, Paraclostridium bifermentans with CdS nanoparticles, Journal of Environmental Chemical Engineering, J. Environ. Chem. Eng. 11(5),110364.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, D., Liu, C., Luo, R., Sadakane, K., Lam, T., 2015. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics 31(10), 1674\u0026ndash;1676.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLong, M., Zhou, C., Xia, S., Guadiea, A., 2017. Concomitant Cr(VI) reduction and Cr(III) precipitation with nitrate in a methane/oxygen-based membrane biofilm reactor. Chem. Eng. J. 315, 58\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagoc, T., Salzberg, S.L., 2011. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27(21), 2957\u0026ndash;2963.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMin, XY., Zhang, KJ., Jianxin Chen, Liyuan Chai, Zhang Lin, Long Zou, Weizao Liu, Chunlian Ding, Yan Shi., 2024. Bacteria-driven copper redox reaction coupled electron transfer from Cr(VI) to Cr(III): A new and alternate mechanism of Cr(VI) bioreduction. J HAZARD MATER. 462, 132485.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeal, A.L., Clough, L.K., Perkins, T.D., Little, B.J., Magnuson, T.S., 2004. In situ measurement of Fe(III) reduction activity of \u003cem\u003eGeobacter pelophilus\u003c/em\u003e by simultaneous in situ RT-PCR and XPS analysis. FEMS Microbiol. Ecol. 49(1), 163\u0026ndash;169.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNevin, K.P., Holmes, D.E., Woodard, T.L., Hinlein, E.S., Ostendorf, D.W., Lovley, D.R., 2005. Geobacter bemidjiensis sp. nov. and Geobacter psychrophilus sp. nov., two novel Fe(III)-reducing subsurface isolates. Int J Syst Evol Microbiol 55(Pt 4), 1667\u0026ndash;1674.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePanda, J., Sarkar, P., 2012. Bioremediation of chromium by novel strains Enterobacter aerogenes T2 and Acinetobacter sp. PD 12 S2. Environ. Sci. Pollut. R. 19(5), 1809\u0026ndash;1817.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParks, D.H., Imelfort, M., Skennerton, C.T., Hugenholtz, P., Tyson, G.W., 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 25(7), 1043\u0026ndash;1055.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., Gl\u0026ouml;ckner, F.O., 2012. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41(D1), D590-D596.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahman, A., Nahar, N., Nawani, N.N., Jass, J., Hossain, K., Saud, Z.A., Saha, A.K., Ghosh, S., Olsson, B., Mandal, A., 2015. Bioremediation of hexavalent chromium (VI) by a soil-borne bacterium, Enterobacter cloacae B2-DHA. J Environ Sci Health A Tox Hazard Subst Environ Eng 50(11), 1136\u0026ndash;1147.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahman, A., Nahar, N., Nawani, N.N., Jass, J., Hossain, K., Saud, Z.A., Saha, A.K., Ghosh, S., Olsson, B., Mandal, A., 2015. Bioremediation of hexavalent chromium (VI) by a soil-borne bacterium, Enterobacter cloacae B2-DHA. Journal of environmental science and health. Part A, Toxic/hazardous substances \u0026amp; environmental engineering 50(11), 1136\u0026ndash;1147.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahman, Z., Singh, V.P., 2014. Cr(VI) reduction by Enterobacter sp. DU17 isolated from the tannery waste dump site and characterization of the bacterium and the Cr(VI) reductase. Int. Biodeter. Biodegr. 91, 97\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahman, Z., Thomas, L., 2021. Chemical-Assisted Microbially Mediated Chromium (Cr) (VI) Reduction Under the Influence of Various Electron Donors, Redox Mediators, and Other Additives: An Outlook on Enhanced Cr(VI) Removal. Front. Microbiol. 11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamirez-Diaz, M.I., Diaz-Perez, C., Vargas, E., Riveros-Rosas, H., Campos-Garcia, J., Cervantes, C., 2008. Mechanisms of bacterial resistance to chromium compounds. Biometals 21(3), 321\u0026ndash;332.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSANI, R.K., PEYTON, B.M., SMITH, W.A., APEL, W.A., PETERSEN, J.N., Idaho, N.L.I., 2002. Dissimilatory reduction of Cr(VI), Fe(III), and U(VI) by Cellulomonas isolates. Appl. Microbiol. Biot. 60(1\u0026ndash;2), 192\u0026ndash;199.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, C., Cui, Y., Lu, J., Zhang, B., 2020. Sulfur-based autotrophic biosystem for efficient vanadium (V) and chromium (VI) reductions in groundwater. Chem. Eng. J. 395, 124972.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, J., Zhang, B., Qiu, R., Lai, C., Jiang, Y., He, C., Guo, J., 2019. Microbial Chromate Reduction Coupled to Anaerobic Oxidation of Elemental Sulfur or Zerovalent Iron. Environ. Sci. Technol. 53(6), 3198\u0026ndash;3207.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSieber, C.M.K., Probst, A.J., Sharrar, A., Thomas, B.C., Hess, M., Tringe, S.G., Banfield, J.F., 2018. Recovery of genomes from metagenomes via a dereplication, aggregation and scoring strategy. Nature Microbiology 3(7), 836\u0026ndash;843.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStraub, K.L., Buchholz-Cleven, B.E., 2001. Geobacter bremensis sp. nov. and Geobacter pelophilus sp. nov., two dissimilatory ferric-iron-reducing bacteria. Int J Syst Evol Microbiol 51(Pt 5), 1805\u0026ndash;1808.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu, X.L., Tian, Q., Zhang, J., Yuan, X.Z., Shi, X.S., Guo, R.B., Qiu, Y.L., 2014. Acetobacteroides hydrogenigenes gen. nov., sp. nov., an anaerobic hydrogen-producing bacterium in the family Rikenellaceae isolated from a reed swamp. Int J Syst Evol Microbiol 64(Pt 9), 2986\u0026ndash;2991.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, Y., Lan, J., Du, Y., Guo, L., Du, D., Chen, S., Ye, H., Zhang, T.C., 2020. Chromium(VI) bioreduction and removal by Enterobacter sp. SL grown with waste molasses as carbon source: Impact of operational conditions. Bioresource Technol. 302, 121974.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamura, K., Stecher, G., Kumar, S., 2021. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 38(7), 3022\u0026ndash;3027.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian, Y., Zhang, H., Zheng, L., Li, S., Hao, H., Yin, M., Cao, Y., Huang, H., 2019. Process Analysis of Anaerobic Fermentation Exposure to Metal Mixtures. Int. J. Env. Res. Pub. He. 16(14), 2458.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViamajala, S., Smith, W.A., Sani, R.K., Apel, W.A., Petersen, J.N., Neal, A.L., Roberto, F.F., Newby, D.T., Peyton, B.M., 2007. Isolation and characterization of Cr(VI) reducing Cellulomonas spp. from subsurface soils: Implications for long-term chromate reduction. Bioresource Technol. 98(3), 612\u0026ndash;622.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViti, C., Pace, A., Giovannetti, L., 2003. Characterization of Cr(VI)-resistant bacteria isolated from chromium-contaminated soil by tannery activity. Curr. Microbiol. 46(1), 1\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVollmers, J., Wiegand, S., Lenk, F., Kaster, A., 2022. How clear is our current view on microbial dark matter? (Re-)assessing public MAG \u0026amp; SAG datasets with MDMcleaner. Nucleic Acids Res. 50(13), e76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, P.C., Mori, T., Komori, K., Sasatsu, M., Toda, K., Ohtake, H., 1989. Isolation and Characterization of an Enterobacter cloacae Strain That Reduces Hexavalent Chromium under Anaerobic Conditions. Appl Environ Microbiol 55(7), 1665\u0026ndash;1669.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, Y., Simmons, B.A., Singer, S.W., 2016. MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets. Bioinformatics 32(4), 605\u0026ndash;607.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia, X., Wu, S., Li, N., Wang, D., Zheng, S., Wang, G., 2018. Novel bacterial selenite reductase CsrF responsible for Se(IV) and Cr(VI) reduction that produces nanoparticles in Alishewanella sp. WH16-1. J. Hazard. Mater. 342, 499\u0026ndash;509.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia, X., Wu, S., Zhou, Z., Wang, G., 2021. Microbial Cd(II) and Cr(VI) resistance mechanisms and application in bioremediation. J. Hazard. Mater. 401, 123685.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan, W., Wang, N., Wei, D., Liang, C., Chen, X., Liu, L., Shi, J., 2021. Bacterial community compositions and nitrogen metabolism function in a cattle farm wastewater treatment plant revealed by Illumina high-throughput sequencing. Environ. Sci. Pollut. R. 28(30), 40895\u0026ndash;40907.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, W., Hong, W., Huang, Y., Li, S., Li, M., Zhong, H., He, Z., 2022. Exploration on the Cr(VI) resistance mechanism of a novel thermophilic Cr(VI)-reducing bacteriaAnoxybacillus flavithermusABF1 isolated from Tengchong geothermal region, China. Env. Microbiol. Rep.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, KC., Zhang, WJ., Zhentian Liang, Hongyu Zhao, Juanfen Chai, Yuesuo Yang, Tingting Teng, Zhang, DY., 2023. Facilitating New Chromium Reducing Microbes to Enhance Hexavalent Chromium Reduction by In Situ Sonoporation-Mediated Gene Transfer in Soils. Environ. Sci. Technol. 57, 40, 15123\u0026ndash;15133.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, B., Wang, Z., Shi, J., Dong, H., 2020. Sulfur-based mixotrophic bio-reduction for efficient removal of chromium (VI) in groundwater. Geochim. Cosmochim. Ac. 268, 296\u0026ndash;309.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, J., Zhou, W., Liu, B., He, J., Shen, Q., Zhao, F., 2015. Anaerobic arsenite oxidation by an autotrophic arsenite-oxidizing bacterium from an arsenic-contaminated paddy soil. Environ. Sci. Technol. 49(10), 5956\u0026ndash;5964.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong, J., Yin, W., Li, Y., Li, P., Wu, J., Jiang, G., Gu, J., Liang, H., 2017. Column study of enhanced Cr(VI) removal and longevity by coupled abiotic and biotic processes using Fe(0) and mixed anaerobic culture. Water Res. 122, 536\u0026ndash;544.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chromate reduction, chromium-reducing bacteria, microbial diversity, microbial mechanism","lastPublishedDoi":"10.21203/rs.3.rs-8418132/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8418132/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHexavalent chromium [Cr(VI)] is a heavy metal that poses serious environmental risk. Microbial Cr(VI) reduction is a potential remediation approach to reduce the mobility and toxicity of this metal in the environment. However, the diversity and metabolic mechanisms of Cr(VI)-reducing bacteria (CrRBs) remain unknown. In this study, a combination of enrichment incubation and high-throughput sequencing was used to elucidate CrRBs and their associated metabolic pathways for Cr(VI) reduction. Enrichment incubation identified bacterial populations belonging to \u003cem\u003eCellulomonas, Enterobacter, Rikenellaceae\u003c/em\u003e, and \u003cem\u003eCitrifermentans\u003c/em\u003e as putative CrRBs in the two Cr(VI)-contaminated sediments and paddy soils as they gradually dominated the microbial community. High-quality metagenome-assembled genomes (MAGs) associated with putative CrRBs were reconstructed, and functional genes responsible for Cr(VI) reduction were detected, suggesting that they are putative CrRBs. This study advances our understanding of CrRBs diversity and their underlying metabolic mechanisms.\u003c/p\u003e","manuscriptTitle":"Deciphering microbial mechanisms for hexavalent chromium reduction in contaminated sediment and paddy soil","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-11 10:17:00","doi":"10.21203/rs.3.rs-8418132/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6262411b-a803-4ab9-a2c4-b3fdb283d8df","owner":[],"postedDate":"March 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-04T16:24:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-11 10:17:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8418132","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8418132","identity":"rs-8418132","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}